1. Field of the Invention
The present invention generally relates to confocal microscopy. More particularly, the invention relates to increasing the scanning rate capability of confocal microscopes.
2. Description of Related Art
Confocal microscopy is a technique that allows visualization of small structures in light scattering material such as brain slices. It accomplishes this by combining point illumination with point detection. The point detection is achieved by using a pinhole in an image plane that serves to filter light from out-of-focus planes above and below the area of interest thereby creating an optical section of a relatively thick specimen.
The main limitation of confocal microscopes is the speed of image acquisition, since every image is reconstructed on a point-by-point basis. Typical commercial systems, which rely on relatively slow galvanometer-driven mirrors to position the point illumination, have frame rates of approximately 1 Hz. Even the fastest systems, which scan several illumination spots simultaneously, can only record at approximately 200 Hz. One way that the slower systems are used for faster recording is by only collecting data from the pixels lying on a single line, but even this line-scan technique, which sacrifices flexibility in picking sites-of-interest, only boosts the effective frame rate to approximately 400 Hz. With the majority of current systems, faster imaging time is directly related to shorter dwell times at each site-of-interest, which reduces the achievable signal-to-noise ratio. To achieve the frame rate necessary for making functional recordings at several user-selected sites-of-interest, it is beneficial to have an addressable system that can selectively visit several sites on a specimen without spending any time scanning over areas that do not contain structures of interest.
The use of acousto-optic deflectors (AODs) can increase the speed at which the point illumination may be positioned and allows for random access scanning at user-selected sites-of-interest. However, the use of AODs necessitates a path of light returning from the specimen that is different than the illumination path and thus prevents the use of a stationary pinhole. This in turn requires a pinhole or filter that is spatially and temporally synchronized with the scanning excitation spot. Although there are existing systems that utilize an AOD, those systems only utilize an AOD to reposition the illumination point in one dimension. The deflection of the illumination point in the second dimension is accomplished by a relatively slow galvanometer-driven mirror such as one used on typical confocal microscopes. In addition, the existing systems that utilize an AOD employ a slit in the direction that the AOD deflects the illumination point, rather than a pinhole, thereby preventing true confocal imaging.
There exists, therefore, a need for a confocal microscope that permits flexibility in selecting sites-of-interest with increased scanning and recording rates for observing high-speed phenomena without reducing dwell time at each site-of-interest. Furthermore, to enable accurate site selection, the same system should be able to collect full frame confocal images.